5-HT2B antagonists

ABSTRACT

The invention provides novel compounds and compositions comprising a 5-HT 2B  antagonist of formula I: 
                         
and related methods for treating a person having a disorder characterized by undesirable 5-HT 2B  receptor signaling, such as migraine, irritable bowel syndrome (IBS), pulmonary arterial hypertension (PAH), fibrosis, hepatocellular cancer, a small intestinal neuroendocrine tumor, cardiovascular disorders, and gastrointestinal (GI) tract disorders.

This application is a continuation of PCT/CN2015/076079; Filed: Apr. 8,2015, which claims priority to PCT/CN2014/075285; Filed: Apr. 14, 2014.

INTRODUCTION

G-protein coupled receptors (GPCRs) are involved in numerousphysiological processes and represent major pharmaceutical targets indrug discovery. Over 40% of marketed drugs act through modulatingGPCRs.¹ In the past, GPCR drugs were mainly discovered based ontraditional medicinal chemistry approach, which restricted GPCR drugs tolimited scaffold space. More recently, docking screens against GPCRcrystal structures have been successfully applied in identification ofnew potent ligands.²⁻⁴

One recently determined GPCR structures is 5-HT_(2B) receptor.^(5,6) Itbelongs to 5-HT₂ receptor family, which is comprised of three subtypes:5-HT_(2A), 5-HT_(2B) and 5-HT_(2C). Although both 5-HT_(2A) and5-HT_(2C) receptors have been widely studied as therapeutic targets,research on 5-HT_(2B) has been limited. 5-HT_(2B) agonism has beenregarded as an off-target since it activation is related to cardiachypertrophy and pulmonary hypertension.^(7,8) Many recent studies havefocused on the possible application of 5-HT_(2B) antagonists.⁹ Severalcompounds have advanced to clinical trials or pre-clinical research astreatments for migraine disorders,¹⁰ irritable bowel syndrome(IBS),^(11,12) pulmonary arterial hypertension (PAH)^(13,14) andfibrosis.¹⁵ Besides, some studies have suggested that 5-HT could promotecell survival and growth of hepatocellular carcinoma (HCC) by activationof 5-HT_(2B) receptor.^(16,17) And our recent research has discoveredthat sorafenib, a kinase drug approved to treat hepatocellularcarcinoma, also binds to 5-HT_(2B) receptor, which indicated that thebinding of 5-HT_(2B) receptor might contribute to the sorafenib-producedanticancer effect.¹⁸ Currently, only few highly selective 5-HT_(2B)antagonists have been reported due to high degree of homology with itsclose members, which hamper the further understanding of the 5-HT_(2B)receptor roles.¹⁹ Therefore, discovery of novel and selective 5-HT_(2B)antagonist would be of great interest to further explore the functionand therapeutic application of 5-HT_(2B) receptor.

Ma et al. (Bioorg. Med. Chem. Lett. 19, 2009, 5644-7) disclose2,4-diamino-1,3,5-triazine derivatives, including related compounds5a-5j, that “could be used as leads for the discovery of neuronal sodiumchannels blockers for managing central nervous system related disorders”(abstract) and provides “a lead molecule for further investigation andoptimization for neuronal sodium channel binding activity, thetherapeutic benefits of which are yet to be established” (finalsentence).

US 2009/0226422A1 discloses Table 3, a list of hundreds of compoundsincluding related compounds 82 and 320(N-4-(4-methoxyphenyl)-1,3,5-triazaspiro[5.5]undeca-1,3-diene-2,4-diamineandN-4-(3-methoxyphenyl)-1,3,5-triazaspiro[5.5]undeca-1,3-diene-2,4-diamine),“identified by E47-ID1 interaction mapping as potentially inhibitingE47-Id1 interaction”, wherein Id1 is an inhibitor of differentiationprotein, and E47 is a ubiquitously expressed transcription factor whichcan bind and be sequestered by Id1. US 2009/0226422A1 teaches nothingfurther about these two compounds, other than they might potentiallyinhibit E47-Id1 interaction, and these compounds are not encompassed bythe compounds alleged to be useful for treating proliferative disorders,i.e. formulas I-IV.

WO2010024225 and WO2012149266 also disclose related compounds.

SUMMARY OF THE INVENTION

The invention provides methods and compositions for treating a personhaving a disorder characterized by over-, or undesirable 5-HT_(2B)receptor signaling, comprising administering to the person a 5-HT_(2B)antagonist of formula I:

wherein:

R¹ and R² are independently H or methyl;

R³ and R⁴ are independently a C1-C4 alkyl, or R³ and R⁴ are joined toform a C3-C8 cycloalkyl; and

R⁵-R⁹ are independently H or an optionally substituted heteroatom(particularly halogen or hydroxyl), or C1-C4 alkyl, C1-C4 alkyloxy,carbonyl, carboxyl, or amine, each of which is optionally substitutedand may optionally comprise 1-3 heteroatoms; or salt thereof.

In embodiments:

R¹ and R² are independently H or methyl;

R³ and R⁴ are independently C1-C3 alkyl, or R³ and R⁴ are joined to formC4-C7 cycloalkyl;

R⁵ and R⁹ are independently H, halogen, methyl or methoxyl; and/or

R⁶-R⁸ are independently H, halogen, methyl, —OR¹⁰, COR¹⁰, COOR¹⁰, orCONR¹⁰R¹⁰, wherein each R¹⁰ is independently H or C1-C4 alkyl.

In further embodiments:

R¹ and R² are independently H or methyl;

R³ and R⁴ are methyl or R³ and R⁴ form cyclopentyl or cyclohexyl;

R⁵ is H, halogen, methyl or methoxyl;

R⁶ is H, halogen (F, Cl, Br, I), methyl, methoxyl, or —OR¹⁰, COR¹⁰,COOR¹⁰, or CONR¹⁰R¹⁰, wherein each R¹⁰ is independently H or C1-C4alkyl.

R⁷ is H, halogen, methyl, —OR¹⁰ or COOR¹⁰, wherein each R¹⁰ isindependently H or C1-C3 alkyl;

R⁸ is H, halogen, methyl or methoxyl; and/or

R⁹ is H or methyl.

In further embodiments the antagonist is of formula:

In embodiments, the disorder is migraine, irritable bowel syndrome(IBS), pulmonary arterial hypertension (PAH), fibrosis, hepatocellularcancer, a small intestinal neuroendocrine tumor, a cardiovasculardisorder, or a gastrointestinal (GI) tract disorder.

In embodiments, the method further comprises the subsequent step ofdetecting a resultant amelioration of the disorder, and/or theantecedent step of diagnosing the disorder, particularly wherein thedisorder is migraine, irritable bowel syndrome (IBS), pulmonary arterialhypertension (PAH), fibrosis, hepatocellular cancer, a small intestinalneuroendocrine tumor, a cardiovascular disorder, or a gastrointestinal(GI) tract disorder.

The invention also provides pharmaceutical compositions comprising asubject 5-HT_(2B) antagonist and a second, different drug indicted for adisorder characterized by over-, or undesirable 5-HT_(2B) receptorsignaling, particularly migraine, irritable bowel syndrome (IBS),pulmonary arterial hypertension (PAH), fibrosis, hepatocellular cancer,a small intestinal neuroendocrine tumor, a cardiovascular disorder, or agastrointestinal (GI) tract disorder.

The invention also provides novel compounds, compositions are relatedmethods, wherein the compound is a 5-HT_(2B) antagonist of formula I:

wherein:

R¹ and R² are independently H or Me;

R³ and R⁴ form cyclohexyl;

R⁵ is H;

R⁶ is COR¹⁰, COOR¹⁰, or CONR¹⁰R¹⁰, wherein each R¹⁰ is independently Hor C1-C3 alkyl;

R⁷ is H or methyl;

R⁸ is H or halogen; and

R⁹ is H; or salt thereof.

In embodiments,

(3-e1) R¹═H, R²═H, R⁶ is Cl, R⁷═H, R⁸═H;

(3-e2) R¹═H, R²═H, R⁶ is Br, R⁷═H, R⁸═H;

(3-f) R¹═H, R²═H, R⁶ is I, R⁷═H, R⁸═H;

(3-j) R¹═H, R²═H, R⁶ is CONHEt, R⁷═H, R⁸═H;

(3-k) R¹═H, R²═H, R⁶ is COOPr, R⁷═H, R⁸═H;

(3-v) R¹═H, R²═H, R⁶ is COOEt, R⁷=Me, R⁸═H;

(3-w) R¹═H, R²═H, R⁶ is COOEt, R⁷=Me, R⁸═Br;

(3-x) R¹=Me, R²═H, R⁶ is COOEt, R⁷═H, R⁸═H;

(3-y) R¹=Me, R²=Me, R⁶ is COOEt, R⁷═H, R⁸═H; or

(3-z) R¹═H, R²═H, R⁶ is COPr, R⁷═H, R⁸═H.

In further embodiments, the novel compound is of formula:

The invention also provides a pharmaceutical composition comprising asubject compound and a pharmaceutically-acceptable excipient, in unitdosage, particularly therapeutically effective unit dosage, wherein thecompound optionally mixed, coformulated or copackaged with a second,different drug indicted for a disorder characterized by over-, orundesirable 5-HT_(2B) receptor signaling, particularly migraine,irritable bowel syndrome (IBS), pulmonary arterial hypertension (PAH),fibrosis, hepatocellular cancer, a small intestinal neuroendocrinetumor, a cardiovascular disorder, or a gastrointestinal (GI) tractdisorder.

The invention encompasses all combination of the particular embodimentsrecited herein.

DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION

The following descriptions of particular embodiments and examples areprovided by way of illustration and not by way of limitation. Thoseskilled in the art will readily recognize a variety of noncriticalparameters that could be changed or modified to yield essentiallysimilar results.

The invention provides methods and compositions for treating a personhaving a disorder characterized by over-, or undesirable 5-HT_(2B)receptor signaling, such as migraine, irritable bowel syndrome (IBS),pulmonary arterial hypertension (PAH), fibrosis, including liverfibrosis, lung fibrosis and pulmonary fibrosis; hepatocellular cancer,small intestinal neuroendocrine tumors, cardiovascular disorders, suchas chronic heart disease, congestive heart failure and hypertension; andgastrointestinal (GI) tract disorders, especially disorders involvingaltered motility, hypertonic lower esophageal sphinter and particularlyIBS (WO 01/08668, WO 2003035646); disorders of gastric motility,dyspepsia, GERD, tachygastria. Additional documented indications includemigraine/neurogenic pain (WO 97/44326); pain (U.S. Pat. No. 5,958,934);anxiety (WO 97/44326); depression (WO 97/44326); benign prostatichyperplasia (U.S. Pat. No. 5,952,331); sleep disorder (WO 97/44326);panic disorder, obsessive compulsive disorder, alcoholism, hypertension,anorexia nervosa, and priapism (WO 96/24351); incontinence and bladderdysfunction (WO 96/24351); disorders of the uterus, such ashysmenorrhoea, pre-term labour, post-partum remodeling, restenosis,asthma and obstructive airway disease (WO 2003035646).

These indications are all supported by convenient functional, clinicaland/or animal activity models, by examples: for migraine (e.g Johnson etal., Cephalalgia, 2003, 23, 117-123), pulmonary hypertension (e.g.Launay et al. 2002, Nat Med 8 (10), 1129-35); IBS (e.g. Borman et al.British J Pharmacol (2002) 135, 1144-51, fibrosis (e.g. Svejda et al.Cancer (Jun. 15, 2010), 2902-12), pulmonary hypertension (e.g. Blanpainet al., Cardiovascular Res 60 (2003) 518-528), liver fibrosis (e.g.Rudell et al. Amer J Pathol, September 2006, 169 (3), 861-76); pulmonaryarterial hypertension and ventricular hypertrophy (e.g. Porvasnik etal., 2010, J Pharmacol and Experimental Therapeutics, 334 (2) 364-72),and chronic liver disease (e.g. Ebrahimkhani et al., December 2011,Nature Med 17 (12), 1668-74).

In embodiments, the subject compounds are administered in conjunctionwith, or mixed, coformulated or copackaged with a second, different drugindicted for a disorder characterized by over-, or undesirable 5-HT_(2B)receptor signaling:

Exemplary Coadministration/Coformulations

Indication Second Drug migraine Pain medications, e.g. NSAID, Ergotalkaloids, triptans irritable bowel Antidiarrheals, bile acid syndrome(IBS) binding agents pulmonary arterial Antihypertensives, e.g.hypertension (PAH) calcium channel blockers fibrosis Immunosuppressants,e.g. corticosteroids hepatocellular cancer Chemotherapies, e.g.oxorubicin, 5-fluorouracil, cisplatin small intestinal Chemotherapies,e.g. neuroendocrine tumor cisplatin, etoposide cardiovascular disorderAntianginals, antiarrythmics, anticoagulants, antihypertensives, betablockers, calcium channel blockers, cardiac glycosides, diuretics,vasodilators gastrointestinal (GI) Anticholinergics, tract disorderantidiarreals, antiemetics, antiulcer medications.

Unless contraindicated or noted otherwise, in these descriptions andthroughout this specification, the terms “a” and “an” mean one or more,the term “or” means and/or and polynucleotide sequences are understoodto encompass opposite strands as well as alternative backbones describedherein. Furthermore, genuses are recited as shorthand for a recitationof all members of the genus; for example, the recitation of (C1-C3)alkyl is shorthand for a recitation of all C1-C3 alkyls: methyl, ethyland propyl, including isomers thereof.

The term “heteroatom” as used herein generally means any atom other thancarbon or hydrogen. Preferred heteroatoms include oxygen (O), phosphorus(P), sulfur (S), nitrogen (N), and halogens, and preferred heteroatomfunctional groups are haloformyl, hydroxyl, aldehyde, amine, azo,carboxyl, cyanyl, thocyanyl, carbonyl, halo, hydroperoxyl, imine,aldimine, isocyanide, iscyante, nitrate, nitrile, nitrite, nitro,nitroso, phosphate, phosphono, sulfide, sulfonyl, sulfo, and sulfhydryl.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which is fully saturated,having the number of carbon atoms designated (i.e. C1-C8 means one toeight carbons). Examples of alkyl groups include methyl, ethyl,n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl and the like.

The term “alkenyl”, by itself or as part of another substituent, means astraight or branched chain, or cyclic hydrocarbon radical, orcombination thereof, which may be mono- or polyunsaturated, having thenumber of carbon atoms designated (i.e. C2-C8 means two to eightcarbons) and one or more double bonds. Examples of alkenyl groupsinclude vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl) and higher homologs and isomersthereof.

The term “alkynyl”, by itself or as part of another substituent, means astraight or branched chain hydrocarbon radical, or combination thereof,which may be mono- or polyunsaturated, having the number of carbon atomsdesignated (i.e. C2-C8 means two to eight carbons) and one or moretriple bonds. Examples of alkynyl groups include ethynyl, 1- and3-propynyl, 3-butynyl and higher homologs and isomers thereof.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from alkyl, as exemplified by—CH₂—CH₂—CH₂—CH₂—. Typically, an alkyl (or alkylene) group will havefrom 1 to 24 carbon atoms, with those groups having 10 or fewer carbonatoms being preferred in the invention. A “lower alkyl” or “loweralkylene” is a shorter chain alkyl or alkylene group, generally havingeight or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and from one to three heteroatoms selectedfrom the group consisting of O, N, P, Si and S, wherein the nitrogen,sulfur, and phosphorous atoms may optionally be oxidized and thenitrogen heteroatom may optionally be quaternized. The heteroatom(s) O,N, P and S may be placed at any interior position of the heteroalkylgroup. The heteroatom Si may be placed at any position of theheteroalkyl group, including the position at which the alkyl group isattached to the remainder of the molecule. Examples include—CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃,—CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃,—CH₂—CH═N—OCH₃, and —CH═CH—N(CH3)-CH₃. Up to two heteroatoms may beconsecutive, such as, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃.

Similarly, the term “heteroalkylene,” by itself or as part of anothersubstituent means a divalent radical derived from heteroalkyl, asexemplified by —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms can also occupy either or both of thechain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Accordingly, acycloalkyl group has the number of carbon atoms designated (i.e., C3-C8means three to eight carbons) and may also have one or two double bonds.A heterocycloalkyl group consists of the number of carbon atomsdesignated and from one to three heteroatoms selected from the groupconsisting of O, N, Si and S, and wherein the nitrogen and sulfur atomsmay optionally be oxidized and the nitrogen heteroatom may optionally bequaternized. Additionally, for heterocycloalkyl, a heteroatom can occupythe position at which the heterocycle is attached to the remainder ofthe molecule. Examples of cycloalkyl include cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include 1-(1,2,5,6-tetrahydropyrid-yl), 1-piperidinyl,2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl,tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl,tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like.

The terms “halo” and “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include alkyl substituted with halogen atoms, which can be thesame or different, in a number ranging from one to (2m′+1), where m′ isthe total number of carbon atoms in the alkyl group. For example, theterm “halo(C1-C4)alkyl” is mean to include trifluoromethyl,2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like. Thus,the term “haloalkyl” includes monohaloalkyl (alkyl substituted with onehalogen atom) and polyhaloalkyl (alkyl substituted with halogen atoms ina number ranging from two to (2m′+1) halogen atoms, where m′ is thetotal number of carbon atoms in the alkyl group). The term“perhaloalkyl” means, unless otherwise stated, alkyl substituted with(2m′+1) halogen atoms, where m′ is the total number of carbon atoms inthe alkyl group. For example the term “perhalo(C1-C4)alkyl” is meant toinclude trifluoromethyl, pentachloroethyl,1,1,1-trifluoro-2-bromo-2-chloroethyl and the like.

The term “acyl” refers to those groups derived from an organic acid byremoval of the hydroxy portion of the acid. Accordingly, acyl is meantto include, for example, acetyl, propionyl, butyryl, decanoyl, pivaloyl,benzoyl and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,typically aromatic, hydrocarbon substituent which can be a single ringor multiple rings (up to three rings) which are fused together or linkedcovalently. Non-limiting examples of aryl groups include phenyl,1-naphthyl, 2-naphthyl, 4-biphenyl and 1,2,3,4-tetrahydronaphthalene.

The term heteroaryl,” refers to aryl groups (or rings) that contain fromzero to four heteroatoms selected from N, O, and S, wherein the nitrogenand sulfur atoms are optionally oxidized and the nitrogen heteroatom areoptionally quaternized. A heteroaryl group can be attached to theremainder of the molecule through a heteroatom. Non-limiting examples ofheteroaryl groups include 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyland 6-quinolyl.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) is meant to include both substituted and unsubstitutedforms of the indicated radical. Preferred substituents for each type ofradical are provided below.

Substituents for the alkyl and heteroalkyl radicals (as well as thosegroups referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl,alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl andheterocycloalkenyl) can be a variety of groups selected from: —OR′, ═O,═NR′, ═N—OR′, —NR′R″, —SR′, halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′,—CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″,—NR′—SO₂NR′″, —NR″CO₂R′, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH, —NH—C(NH₂)═NR′,—S(O)R′, —SO₂R′, —SO₂NR′R″, —NR″SO₂R, —CN and —NO₂, in a number rangingfrom zero to three, with those groups having zero, one or twosubstituents being particularly preferred. R′, R″ and R′″ eachindependently refer to hydrogen, unsubstituted (C1-C8)alkyl andheteroalkyl, unsubstituted aryl, aryl substituted with one to threehalogens, unsubstituted alkyl, alkoxy or thioalkoxy groups, oraryl-(C1-C4)alkyl groups. When R′ and R″ are attached to the samenitrogen atom, they can be combined with the nitrogen atom to form a 5-,6- or 7-membered ring. For example, —NR′R″ is meant to include1-pyrrolidinyl and 4-morpholinyl. Typically, an alkyl or heteroalkylgroup will have from zero to three substituents, with those groupshaving two or fewer substituents being preferred in the invention. Morepreferably, an alkyl or heteroalkyl radical will be unsubstituted ormonosubstituted. Most preferably, an alkyl or heteroalkyl radical willbe unsubstituted. From the above discussion of substituents, one ofskill in the art will understand that the term “alkyl” is meant toinclude groups such as trihaloalkyl (e.g., —CF₃ and —CH₂CF₃).

Preferred substituents for the alkyl and heteroalkyl radicals areselected from: —OR′, ═O, —NR′R″, —SR′, halogen, —SiR′R″R′″, —OC(O)R′,—C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR″CO₂R′,—NR′—SO₂NR″R′″, —S(O)R′, —SO2R′, —SO₂NR′R″, —NR″SO₂R, —CN and —NO₂,where R′ and R″ are as defined above. Further preferred substituents areselected from: —OR′, ═O, —NR′R″, halogen, —OC(O)R′, —CO₂R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR″CO₂R′, —NR′—SO₂NR″R′″, —SO₂R′, —SO₂NR′R″,—NR″SO₂R, —CN and —NO₂.

Similarly, substituents for the aryl and heteroaryl groups are variedand selected from: halogen, —OR′, —OC(O)R′, —NR′R″, —SR′, —R′, —CN,—NO₂, —CO₂R′, —CONR′R″, —C(O)R′, —OC(O)NR′R″, —NR″C(O)R′, —NR″CO2R′,—NR′—C(O)NR″R′″, —NR′—SO₂NR″R′″, —NH—C(NH2)═NH, —NR′C(NH₂)═NH,—NH—C(NH₂)═NR′, —S(O)R′, —SO₂R′, —SO₂NR′R″, —NR″SO₂R, —N₃, —CH(Ph)₂,perfluoro(C1-C4)alkoxy and perfluoro(C1-C4)alkyl, in a number rangingfrom zero to the total number of open valences on the aromatic ringsystem; and where R′, R″ and R′″ are independently selected fromhydrogen, (C1-C8)alkyl and heteroalkyl, unsubstituted aryl andheteroaryl, (unsubstituted aryl)-(C1-C4)alkyl and (unsubstitutedaryl)oxy-(C1-C4)alkyl. When the aryl group is1,2,3,4-tetrahydronaphthalene, it may be substituted with a substitutedor unsubstituted (C3-C7)spirocycloalkyl group. The(C3-C7)spirocycloalkyl group may be substituted in the same manner asdefined herein for “cycloalkyl”. Typically, an aryl or heteroaryl groupwill have from zero to three substituents, with those groups having twoor fewer substituents being preferred in the invention. In oneembodiment of the invention, an aryl or heteroaryl group will beunsubstituted or monosubstituted. In another embodiment, an aryl orheteroaryl group will be unsubstituted.

Preferred substituents for aryl and heteroaryl groups are selected from:halogen, —OR′, —OC(O)R′, —NR′R″, —SR′, —R′, —CN, —NO₂, —CO₂R′, —CONR′R″,—C(O)R′, —OC(O)NR′R″, —NR″C(O)R′, —S(O)R′, —SO₂R′, —SO₂NR′R″, —NR″SO₂R,—N₃, —CH(Ph)₂, perfluoro(C1-C4)alkoxy and perfluoro(C1-C4)alkyl, whereR′ and R″ are as defined above. Further preferred substituents areselected from: halogen, —OR′, —OC(O)R′, —NR′R″, —R′, —CN, —NO₂, —CO₂R′,—CONR′R″, —NR″C(O)R′, —SO₂R′, —SO₂NR′R″, —NR″SO₂R,perfluoro(C1-C4)alkoxy and perfluoro(C1-C4)alkyl.

The substituent —CO₂H, as used herein, includes bioisostericreplacements therefor; see, e.g., The Practice of Medicinal Chemistry;Wermuth, C. G., Ed.; Academic Press: New York, 1996; p. 203.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CH₂)q-U—, wherein T and U are independently —NH—, —O—, —CH₂— ora single bond, and q is an integer of from 0 to 2. Alternatively, two ofthe substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula -A-(CH2)r-B—,wherein A and B are independently —CH₂—, —O—, —NH—, —S—, —S(O)—,—S(O)₂—, —S(O)₂NR′— or a single bond, and r is an integer of from 1 to3. One of the single bonds of the new ring so formed may optionally bereplaced with a double bond. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula —(CH₂)s-X—(CH₂)t-, where s and t areindependently integers of from 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—,—S(O)₂—, or —S(O)₂NR′—. The substituent R′ in —NR′— and —S(O)₂NR′— isselected from hydrogen or unsubstituted (C1-C6)alkyl.

Preferred substituents are disclosed herein and exemplified in thetables, structures, examples, and claims, and may be applied acrossdifferent compounds of the invention, i.e. substituents of any givencompound may be combinatorially used with other compounds.

In particular embodiments applicable substituents are independentlysubstituted or unsubstituted heteroatom, substituted or unsubstituted,optionally heteroatom C1-C6 alkyl, substituted or unsubstituted,optionally heteroatom C2-C6 alkenyl, substituted or unsubstituted,optionally heteroatom C2-C6 alkynyl, or substituted or unsubstituted,optionally heteroatom C6-C14 aryl, wherein each heteroatom isindependently oxygen, phosphorus, sulfur or nitrogen.

In more particular embodiments, applicable substituents areindependently aldehyde, aldimine, alkanoyloxy, alkoxy, alkoxycarbonyl,alkyloxy, alkyl, amine, azo, halogens, carbamoyl, carbonyl, carboxamido,carboxyl, cyanyl, ester, halo, haloformyl, hydroperoxyl, hydroxyl,imine, isocyanide, iscyante, N-tert-butoxycarbonyl, nitrate, nitrile,nitrite, nitro, nitroso, phosphate, phosphono, sulfide, sulfonyl, sulfo,sulfhydryl, thiol, thiocyanyl, trifluoromethyl or trifluromethyl ether(OCF3).

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds which are prepared with relatively nontoxicacids or bases, depending on the particular substituents found on thecompounds described herein. When compounds of the invention containrelatively acidic functionalities, base addition salts can be obtainedby contacting the neutral form of such compounds with a sufficientamount of the desired base, either neat or in a suitable inert solvent.Examples of pharmaceutically acceptable base addition salts includesodium, potassium, calcium, ammonium, organic amino, or magnesium salt,or a similar salt. When compounds of the invention contain relativelybasic functionalities, acid addition salts can be obtained by contactingthe neutral form of such compounds with a sufficient amount of thedesired acid, either neat or in a suitable inert solvent. Examples ofpharmaceutically acceptable acid addition salts include those derivedfrom inorganic acids like hydrochloric, hydrobromic, nitric, carbonic,monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived fromrelatively nontoxic organic acids like acetic, propionic, isobutyric,oxalic, maleic, malonic, benzoic, succinic, suberic, fumaric, mandelic,phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric,methanesulfonic, and the like. Also included are salts of amino acidssuch as arginate and the like, and salts of organic acids likeglucuronic or galactunoric acids and the like. Certain specificcompounds of the invention contain both basic and acidic functionalitiesthat allow the compounds to be converted into either base or acidaddition salts.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the invention.

In addition to salt forms, the invention provides compounds which are ina prodrug form. Prodrugs of the compounds described herein are thosecompounds that undergo chemical changes under physiological conditionsto provide the compounds of the invention. Additionally, prodrugs can beconverted to the compounds of the invention by chemical or biochemicalmethods in an ex vivo environment. For example, prodrugs can be slowlyconverted to the compounds of the invention when placed in a transdermalpatch reservoir with a suitable enzyme or chemical reagent. Prodrugs areoften useful because they may be easier to administer than the parentdrug, may be more bioavailable by oral administration than the parentdrug, and or may have improved solubility in pharmacologicalcompositions over the parent drug. A wide variety of prodrug derivativesare known in the art, such as those that rely on hydrolytic cleavage oroxidative activation of the prodrug. An example, without limitation, ofa prodrug would be a compound of the invention which is administered asan ester (the “prodrug”), but then is metabolically hydrolyzed to thecarboxylic acid, the active entity.

Certain compounds of the invention can exist in unsolvated forms as wellas solvated forms, including hydrated forms. In general, the solvatedforms are equivalent to unsolvated forms and are intended to beencompassed within the scope of the invention. Certain compounds of theinvention may exist in multiple crystalline or amorphous forms. Ingeneral, all physical forms are equivalent for the uses contemplated bythe invention and are intended to be within the scope of the invention.

Some of the subject compounds possess asymmetric carbon atoms (opticalcenters) or double bonds; the racemates, diastereomers, geometricisomers and specifically designated or depicted chirality is preferredand in many cases critical for optimal activity; however all suchisomers are all intended to be encompassed within the scope of theinvention.

The compounds of the invention may also contain unnatural proportions ofatomic isotopes at one or more of the atoms that constitute suchcompounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example tritium (³H), iodine-125(¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compounds ofthe invention, whether radioactive or not, are intended to beencompassed within the scope of the invention.

The term “therapeutically effective amount” refers to the amount of thesubject compound that will elicit, to some significant extent, thebiological or medical response of a tissue, system, animal or human thatis being sought by the researcher, veterinarian, medical doctor or otherclinician, such as when administered, is sufficient to preventdevelopment of, or alleviate to some extent, one or more of the symptomsof the condition or disorder being treated. The therapeuticallyeffective amount will vary depending on the compound, the disease andits severity and the age, weight, etc., of the mammal to be treated.

The invention also provides pharmaceutical compositions comprising thesubject compounds and a pharmaceutically acceptable excipient,particularly such compositions comprising a unit dosage of the subjectcompounds, particularly such compositions copackaged with instructionsdescribing use of the composition to treat an applicable disease orcondition (herein).

The compositions for administration can take the form of bulk liquidsolutions or suspensions, or bulk powders. More commonly, however, thecompositions are presented in unit dosage forms to facilitate accuratedosing. The term “unit dosage forms” refers to physically discrete unitssuitable as unitary dosages for human subjects and other mammals, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect, in association with asuitable pharmaceutical excipient. Typical unit dosage forms includeprefilled, premeasured ampules or syringes of the liquid compositions orpills, tablets, capsules, losenges or the like in the case of solidcompositions. In such compositions, the compound is usually a minorcomponent (from about 0.1 to about 50% by weight or preferably fromabout 1 to about 40% by weight) with the remainder being variousvehicles or carriers and processing aids helpful for forming the desireddosing form.

Suitable excipients or carriers and methods for preparing administrablecompositions are known or apparent to those skilled in the art and aredescribed in more detail in such publications as Remington'sPharmaceutical Science, Mack Publishing Co, NJ (1991). In addition, thecompounds may be advantageously used in conjunction with othertherapeutic agents as described herein or otherwise known in the art,particularly other anti-necrosis agents. Hence the compositions may beadministered separately, jointly, or combined in a single dosage unit.

The amount administered depends on the compound formulation, route ofadministration, etc. and is generally empirically determined in routinetrials, and variations will necessarily occur depending on the target,the host, and the route of administration, etc. Generally, the quantityof active compound in a unit dose of preparation may be varied oradjusted from about 1, 3, 10 or 30 to about 30, 100, 300 or 1000 mg,according to the particular application. In a particular embodiment,unit dosage forms are packaged in a multipack adapted for sequentialuse, such as blisterpack, comprising sheets of at least 6, 9 or 12 unitdosage forms. The actual dosage employed may be varied depending uponthe requirements of the patient and the severity of the condition beingtreated. Determination of the proper dosage for a particular situationis within the skill of the art. Generally, treatment is initiated withsmaller dosages which are less than the optimum dose of the compound.Thereafter, the dosage is increased by small amounts until the optimumeffect under the circumstances is reached. For convenience, the totaldaily dosage may be divided and administered in portions during the dayif desired.

The compounds can be administered by a variety of methods including, butnot limited to, parenteral, topical, oral, or local administration, suchas by aerosol or transdermally, for prophylactic and/or therapeutictreatment. Also, in accordance with the knowledge of the skilledclinician, the therapeutic protocols (e.g., dosage amounts and times ofadministration) can be varied in view of the observed effects of theadministered therapeutic agents on the patient, and in view of theobserved responses of the disease to the administered therapeuticagents.

The therapeutics of the invention can be administered in atherapeutically effective dosage and amount, in the process of atherapeutically effective protocol for treatment of the patient. Formore potent compounds, microgram (ug) amounts per kilogram of patientmay be sufficient, for example, in the range of about 1, 10 or 100 ug/kgto about 0.01, 0.1, 1, 10, or 100 mg/kg of patient weight though optimaldosages are compound specific, and generally empirically determined foreach compound.

In general, routine experimentation in clinical trials will determinespecific ranges for optimal therapeutic effect, for each therapeutic,each administrative protocol, and administration to specific patientswill also be adjusted to within effective and safe ranges depending onthe patient condition and responsiveness to initial administrations.However, the ultimate administration protocol will be regulatedaccording to the judgment of the attending clinician considering suchfactors as age, condition and size of the patient as well as compoundspotency, severity of the disease being treated. For example, a dosageregimen of the compounds can be oral administration of from 10 mg to2000 mg/day, preferably 10 to 1000 mg/day, more preferably 50 to 600mg/day, in two to four (preferably two) divided doses. Intermittenttherapy (e.g., one week out of three weeks or three out of four weeks)may also be used.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein, including citations therein, are herebyincorporated by reference in their entirety for all purposes.

Examples

We set out to identify new potent 5-HT_(2B) antagonists with novelscaffold and high selectivity via structure-based virtual screening of alarge compound database against 5-HT_(2B) crystal structure. First, werefined the binding site conformations to incorporate knowledge ofantagonist-bound induced-fit effects and assessed the quality of thesemodels in retrospective virtual screening. We also analyzed the bindingsite solvent property to derive several interaction patterns asstructural filters. Then we adopted a hierarchical strategy integratingdifferent computational methods to screen 5-HT_(2B) antagonists from ourin-house compound library. It resulted in 169 candidates meeting thestructural and energetic criteria from more than 100,000 diversedrug-like compounds. Among them we identified molecules with an enrichedscaffold and which adopt a common binding mode. We validated identifiedmolecules in bioassays. We further improved the binding pose andsystematically probed the binding characteristics by testing series ofstructural modifications. The obtained structure-activity relationship(SAR) results are consistent with our binding model. Our scaffold alsoexhibits high selectivity over other 5-HT receptors. Our novel scaffoldprovides 5-HT_(2B) antagonists with improved efficacy and highselectivity.

Synthesis

Step 1: Synthesis of ethyl3-(2,4-diamino-1,3,5-triazaspiro[5.5]undeca-2,4-dien-1-yl)benzoate (1)

A mixture of ethyl 3-aminobenzoate (1.65 g, 10 mmol), cyanoguanidine(925 mg, 11 mmol), conc.HCl (0.83 mL, 10 mmol) and cyclohexanone (981mg, 10 mmol) in EtOH (1.5 mL) was refluxed. After the completion of thereaction, the solid was filtered to obtain compound 1 (1.2 g, 36%).1H-NMR (400 MHz, DMSO-d6) δ (ppm) 9.12 (s, 1H), 8.12-8.06 (m, 1H), 7.81(s, 1H), 7.74-7.56 (m, 4H), 6.43 (s, 1H), 4.45-4.26 (m, 2H), 1.91 (d,J=12.6 Hz, 2H), 1.81-1.43 (m, 5H), 1.43-1.12 (m, 5H), 1.12-0.85 (m, 1H);MS [MH]+ calcd for C17H24N5O2 330.19. found 330.20.

Step 2: Synthesis of ethyl3-(4-amino-1,3,5-triazaspiro[5.5]undeca-2,4-dien-2-ylamino)benzoate

The compound 1 (500 mg, 1.5 mmol) was dissolved in pyridine (2 mL) andEtOH (3 mL), and then the mixture was heated to 120° C. overnight. Thenit was concentrated in vacuo to dryness. After water (10 mL) was added,the mixture was stirred at room temperature for 30 min. Then the solidwas filtered and dried in vacuo to afford the desired product (220 mg,44%). 1H-NMR (400 MHz, DMSO-d6) δ (ppm) 10.40 (s, 1H), 9.03 (d, J=7.2Hz, 2H), 8.02 (d, J=9.1 Hz, 1H), 7.88 (s, 1H), 7.68 (d, J=7.7 Hz, 1H),7.48 (t, J=7.9 Hz, 1H), 7.27 (s, 1H), 4.33 (q, J=7.1 Hz, 2H), 1.76-1.62(m, 8H), 1.50-1.27 (m, 5H); MS [MH]+ calcd for C17H24N5O2 330.19. found330.20.

Step 1: Synthesis of 3-(tert-butoxycarbonylamino) benzoic acid (1)

3-aminobenzoic acid (4.4 g, 32.1 mmol) and di-tert-butyl dicarbonate(10.5 g, 48.2 mmol) were dissolved in anhydrous THF (60 mL). ThenN-ethyl-N-isopropylpropan-2-amine (8.3 g, 64.2 mmol) was added. Thereaction mixture was stirred at room temperature. After the completionof the reaction, it was concentrated in vacco and the residue wasextracted with ethyl acetate. The organic layer was concentrated and theresidue was purified by column chromatograph to provide the desiredproduct (6.2 g, 82%); MS [MH]− calcd for C12H14NO4 236.10. found 236.10.

Step 2: Synthesis of tert-butyl 3-(ethylcarbamoyl) phenylcarbamate (2)

A mixture of compound 1 (2 g, 8.43 mmol), HATU (6.4 g, 16.9 mmol),N-ethyl-N-isopropylpropan-2-amine (3.27 g, 25.3 mmol) was dissolved inDMF (40 mL), and then the mixture was stirred at room temperature for 30min. Then ethanamine hydrochloride (2.1 g, 25.3 mmol) was added. Afterthe completion of the reaction, water was added and the mixture wasextracted with EtOAc for three times. The organic layer was concentratedto give the crude product (3.24 g, 100%) which was used in the next stepwithout further purification.

Step 3: Synthesis of 3-amino-N-ethylbenzamide (3)

Compound 2 (3.24 g, 12.3 mmol) was dissolved in dioxane (40 mL), andthen conc.HCl (10 mL) was added. The mixture was stirred at roomtemperature for 8 h. After the completion of the reaction, it wasconcentrated in vacuo and the residue was purified by columnchromatography (silica gel, PE:EA=4:1) to give the desired product (1.4g, 70%); MS [MH]+ calcd for C9H13N2O 165.09. found 165.10.

Step 4: Synthesis of3-(2,4-diamino-1,3,5-triazaspiro[5.5]undeca-2,4-dien-1-yl)-N-ethylbenzamide(4)

A mixture of compound 3 (1 g, 6.1 mmol), cyanoguanidine (563 mg, 6.7mmol), conc.HCl (0.51 mL, 6.1 mmol) and cyclohexanone (599 mg, 6.1 mmol)in EtOH (2 mL) was refluxed. After the completion of the reaction, thesolid was filtered to give the desired product (1.18 g, 59%) as a whitesolid; MS [MH]+ calcd for C17H25N6O 329.20. found 329.20.

Step 5: Synthesis of3-(4-amino-1,3,5-triazaspiro[5.5]undeca-2,4-dien-2-ylamino)-N-ethylbenzamide

The compound of intermediate 4 (300 mg, 0.91 mmol) was dissolved inpyridine (3 mL), the mixture was heated to 120° C. overnight, then itwas concentrated in vacuo to give the crude product as a salt. Then thesolid was neutralized by sat.NaHCO3 to pH=8. The solid was filtered anddried in vacuo to give the desired product (232 mg, 77%) as a pale pinksolid. 1H-NMR (400 MHz, DMSO-d6) δ (ppm) 10.17 (s, 1H), 8.96 (d, J=14.2Hz, 2H), 8.48 (t, J=5.3 Hz, 1H), 7.78 (d, J=9.3 Hz, 2H), 7.57 (d, J=7.7Hz, 1H), 7.41 (t, J=7.8 Hz, 1H), 7.18 (s, 1H), 3.32-3.23 (m, 2H),1.79-1.33 (m, 10H), 1.12 (t, J=7.2 Hz, 3H); MS [MH]+ calcd for C17H25N6O329.20. found 329.20.

Step 1: Synthesis of 1-cyano-3-methyl-guanidine (1)

Sodium dicyanoamide (5 g, 56.2 mmol) and methylamine hydrochloride (3.8g, 56.2 mmol) were dissolved in n-butanol (25 mL) and H2O (10 mL). Thenthe mixture was refluxed. After the completion of the reaction, themixture was concentrated in vacuo and the residue was purified furtherby column chromatography (silica gel, DCM:MeOH=10:1) to give the desiredproduct compound 1 (1.7 g, 31%); MS [MH]+ calcd for C3H7N4 99.06. found99.10.

Step 2: Synthesis of ethyl3-(3-(N-methylcarbamimidoyl)guanidino)benzoate (2)

A mixture of compound 1 (500 mg, 5.1 mmol), ethyl 3-aminobenzoate (765mg, 4.63 mmol), conc.HCl (0.39 mL, 4.63 mmol) in EtOH (2 mL) wasrefluxed for 6 h. Then the desired product compound 2 was obtained byfiltration (510 mg, 37%); MS [MH]+ calcd for C12H18N5O2 264.14. found264.14.

Step 3: Synthesis of ethyl3-(4-(methylamino)-1,3,5-triazaspiro[5.5]undeca-2,4-dien-2-ylamino)benzoate

Compound of intermediate 2 (510 mg, 1.7 mmol) in cyclohexanone (15 mL)and EtOH (5 mL) was refluxed overnight. Then it was cooled to roomtemperature. Sat. aq. NaHCO3 (25 mL) was added. The mixture wasextracted by DCM (50 mL*3) and washed by brine (30 mL*1). The organiclayers were combined and concentrated in vacuo to afford a crudeproduct, which was purified by column chromatography (silica gel,DCM:MeOH=30:1 to 20:1) to provide the desired product compound 3 (230mg, 39%). 1H-NMR (400 MHz, DMSO-d6) δ (ppm) 8.90 (s, 1H), 7.97 (s, 1H),7.76 (d, J=7.9 Hz, 1H), 7.34 (d, J=7.6 Hz, 1H), 7.23 (t, J=7.8 Hz, 1H),6.42 (s, 1H), 5.77 (s, 1H), 4.27 (q, J=7.1 Hz, 2H), 2.73 (d, J=4.2 Hz,3H), 1.81-1.26 (m, 12H); MS [MH]+ calcd for C18H26N5O2 344.20. found344.20.

Antagonist Activity Assay

Stable cell line, CHO-K1/5-HT_(2B), was applied for the cellularscreening of the compound exhibited significant activities in 5-HT_(2B)antagonist assay using FLIPR method. Briefly, CHO-K1 cells expressing5-HT_(2B) were seeded in a 384-well black-wall, clear-bottom plate at adensity of 20,000 cells per well in 20 μL of growth medium (10% dialyzedFBS+90% F12), 18 hours prior to the day of experiment and maintained at37° C./5% CO2. 20 μL of dye-loading solution and 10 μL of testedcompound solution (at concentrations five times to the final assayconcentrations) were added into the well. Then the plate was placed intoa 37° C. incubator for 60 minutes, followed by 15 minutes at roomtemperature. At last, 12.5 μL of control agonist (at concentrations fivetimes to the EC₈₀ concentrations) was added. The control agonist wasadded to reading plate at 20 s and the fluorescence signal was monitoredfor an additional 100 s (21 s to 120 s). In screening, cells stimulatedwith assay buffer (HBSS-HEPES) containing 0.1% DMSO were chosen asbackground; cell stimulated with 12 nM (EC₈₀ of the cell line) of 5-HTwere chosen as the agonist control; cell treated with SB206553 werechosen as positive control of the screening.

Data acquisition and analyses are performed using ScreenWorks (version3.1) program. The average fluorescent intensity value during 1 s to 20 scalculated as the baseline reading. The relative fluorescent units(ΔRFU) intensity values were calculated with the maximal fluorescentunits (21 s to 120 s) subtracting the average value of baseline reading.The % inhibition of the test article is calculated from the followingequation:%inhibition=[1−(ΔRFU_(Compound)−ΔRFU_(Background))/(ΔRFU_(Agonist control)−ΔRFU_(Background))]*100

Antagonist Activity Tested on 5-HT2B Receptors No. Structure %Inhibition at 1 μM IC₅₀ (nM) 1-a 

<50.0 1-b 

<50.0 1-c 

75.9 ± 11.5 327.6 1-d 

93.1 ± 4.2  1636 1-e 

<50.0 1-f 

<50.0 2508 1-g 

<50.0 1-h 

<50.0 1-i 

<50.0 2-a 

63.4 ± 15.1 1056 2-b 

<50.0 2-c 

<50.0 2-d 

<50.0 3-a 

<50.0 3-b 

<50.0 3-c 

<50.0 3-d 

90.9 ± 2.9  3-e1

95.5 (est) 3-e2

97.4 ± 2.2  33.35 3-f 

101.8 ± 4.5  3-g 

84.6 ± 1.2  280.0 3-h 

<50.0 3-i 

107.2 ± 0.6  27.30 3-j 

95.5 ± 4.8  104.0 3-k 

110.5 ± 2.7  18.10 3-l 

<50.0 3-m 

50.2 ± 8.0  485.9 3-n 

57.2 ± 5.4  531.0 3-o 

<50.0 3-p 

108.6 ± 04.2  3-q 

<50.0 3-r 

66.5 ± 2.1  3-s 

95.7 ± 1.9  177.0 3-t 

69.1 ± 2.7  476.0 3-u 

81.1 ± 6.7  622.4 3-v 

107.5 ± 1.5  3-w 

92.8 ± 3.1  3-x 

107.8 ± 0.0  49.39 3-y 

91.8 ± 0.4  284.5 3-z 

106.0 ± 0.8  35.83

REFERENCES

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What is claimed is:
 1. A method for treating a person in need thereofhaving a disorder characterized by undesirable 5-HT_(2B) receptorsignaling, comprising administering to the person a 5-HT_(2B) antagonistof formula I:

wherein: R¹ and R² are independently H or methyl; R³ and R⁴ areindependently a C1-C4 alkyl, or R³ and R⁴ are joined to form a C3-C8cycloalkyl; and R⁵-R⁹ are independently H, halogen, hydroxyl, or anoptionally substituted C1-C4 alkyl, C1-C4 alkyoxy, carbonyl, carboxyl,or amine; or salt thereof, wherein the disorder is migraine, pulmonaryarterial hypertension (PAH), fibrosis, or a gastrointestinal (GI) tractdisorder selected from the group consisting of hypertonic loweresophageal sphinter, irritable bowel syndrome (IBS), gastric motilitydisorder, dyspepsia, gastroesophageal reflux disease (GERD), andtachygastria.
 2. The method of claim 1 wherein: R¹ and R² areindependently H or methyl; R³ and R⁴ are independently C1-C3 alkyl, orR³ and R⁴ are joined to form C4-C7 cycloalkyl; R⁵ and R⁹ areindependently H, halogen, methyl or methoxyl; and R⁶-R⁸ areindependently H, halogen, methyl, —OR¹⁰, COR¹⁰, COOR¹⁰, or CONR¹⁰R¹⁰,wherein each R¹⁰ is independently H or C1-C4 alkyl.
 3. The method ofclaim 1 wherein: R¹ and R² are independently H or methyl; R³ and R⁴ aremethyl or R³ and R⁴ form cyclopentyl or cyclohexyl; R⁵ is H, halogen,methyl or methoxyl; R⁶ is H, halogen (F, Cl, Br, I), methyl, methoxyl,or —OR¹⁰, COR¹⁰, COOR¹⁰, or CONR¹⁰R¹⁰, wherein each R¹⁰ is independentlyH or C1-C4 alkyl; R⁷ is H, halogen, methyl, —OR¹⁰ or COOR¹⁰, whereineach R¹⁰ is independently H or C1-C3 alkyl; R⁸ is H, halogen, methyl ormethoxyl; and R⁹ is H or methyl.
 4. The method of claim 1 wherein theantagonist is of formula:


5. The method of claim 1 wherein: R¹ and R² are independently H or Me;R³ and R⁴ form cyclohexyl; R⁵ is H; R⁶ is COR¹⁰, COOR¹⁰, or CONR¹⁰R¹⁰,wherein each R¹⁰ is independently H or C1-C4 alkyl; R⁷ is H or methyl;R⁸ is H or halogen; and R⁹ is H; or salt thereof.
 6. The method of claim1 wherein the antagonist is of formula:


7. The method of claim 1 wherein the antagonist is of formula:


8. The method of claim 1 further comprising the step of detecting aresultant amelioration of the disorder.
 9. The method of claim 1 furthercomprising the antecedent step of diagnosing the disorder.
 10. Themethod of claim 1 further comprising administering to the person asecond, different drug indicted for the disorder.
 11. The method ofclaim 1 further comprising administering to the person a second,different drug indicted for the disorder, wherein the second drug anddisorder are selected from the following combinations: INDICATION SECONDDRUG migraine pain medications irritable bowel antidiarrheals, bile acidsyndrome (IBS) binding agents pulmonary arterial antihypertensiveshypertension (PAH) fibrosis immunosuppressants hepatocellular cancerchemotherapies small intestinal chemotherapies neuroendocrine tumorcardiovascular antianginals, disorder antiarrythmics, anticoagulants,antihypertensives, beta blockers, calcium channel blockers, cardiacglycosides, diuretics, vasodilators gastrointestinal (GI)anticholinergics, antidiarreals, tract disorder antiemetics, antiulcermedications.


12. The method of claim 1 wherein the disorder is migraine.
 13. Themethod of claim 1 wherein the disorder is irritable bowel syndrome(IBS).
 14. The method of claim 1 wherein the disorder is pulmonaryarterial hypertension (PAH).
 15. The method of claim 1 wherein thedisorder is fibrosis.
 16. The method of claim 1 wherein the disorder isdyspepsia.
 17. The method of claim 1 wherein the disorder isgastroesophageal reflux disease (GERD).
 18. The method of claim 7wherein the disorder is irritable bowel syndrome (IBS).
 19. The methodof claim 7 wherein the disorder is pulmonary arterial hypertension(PAH).
 20. The method of claim 7 wherein the disorder is fibrosis.